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Overexpression of an nsLTPs-like antimicrobial protein gene (LJAMP2) from motherwort (Leonurus japonicus) enhances resistance to Sclerotinia sclerotiorum in oilseed rape (Brassica napus)
Physiological and Molecular Plant Pathology 82 (2013) 81e87
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Overexpression of an nsLTPs-like antimicrobial protein gene (LJAMP2) from motherwort (Leonurus japonicus) enhances resistance to Sclerotinia sclerotiorum in oilseed rape (Brassica napus) Yuanzhong Jiang a, Xialan Fu a, Mengling Wen a, Fei Wang a, Qiao Tang a, Qiaoyan Tian a, Keming Luo a, b, * a
State Key Laboratory of Eco-Environment and Bio-Resource of Three Gorges Reservoir Region, Key Laboratory of Eco-Environments of Three Gorges Reservoir Region, Ministry of Education, Institute of Resources Botany, School of Life Sciences, Southwest University, Chongqing 400715, China Key Laboratory of Biotechnology and Crop Quality Improvement of Ministry of Agriculture of China, Biotechnology Research Center, Southwest University, Chongqing 400715, China
b
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 3 November 2012
Sclerotinia stem rot caused by Sclerotinia sclerotiorum is one of the most important diseases of oilseed rape worldwide and leads to considerable yield losses. In this study, a non-specific lipid transfer proteinlike antimicrobial protein gene (LJAMP2) from motherwort (Leonurus japonicus) was introduced into oilseed rape (Zhongyou 821) by Agrobacterium-mediated transformation. In vitro experiments revealed that the mycelial growth of S. sclerotiorum was significantly inhibited when supplied with crude leaf extracts from transgenic oilseed rape plants overexpressing LJAMP2. Furthermore, in vivo studies showed that transgenic LJAMP2 plants had enhanced resistance to S. sclerotiorum. Semi-quantitative RT-PCR analysis showed that the LJAMP2 gene was transcribed in all transformed plants. In addition, we also found that overexpression of LJAMP2 in transgenic plants caused constitutive activation of the defenserelated gene PR-1 and an increase of H2O2 production, but did not enhance PDF1.2 expression. Our results suggest that constitutive expression of the LJAMP2 gene from motherwort seeds might be exploited to improve the resistance of oilseed rape against S. sclerotiorum. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Sclerotinia sclerotiorum Antimicrobial protein Oilseed rape LJAMP2
1. Introduction Oilseed rape (Brassica napus L.) is the third largest oilseed crop in the world and provides approximately 13% of the world’s supply of vegetable oil [1]. However, oilseed rape can be seriously infected by Sclerotinia sclerotiorum, which causes the rotting of stems and leaves, resulting in tremendous yield loss worldwide [2]. Due to the lack of endogenous resistance resources in cruciferous plants, little progress has been achieved in the breeding of Sclerotinia resistance in oilseed rape by applying traditional breeding methods [3]. Chemical control of S. sclerotiorum is expensive, often ineffective, and causes environmental pollution. Therefore, the transgenic modification of the crop shows promise for the production of new oilseed rape lines with greater resistance to S. sclerotiorum. S. sclerotiorum, as a necrotrophic fungal pathogen that occurs worldwide, is pathogenic to more than 400 plant species [4,5]. Chitinase and b-1,3-glucanase are capable of catalyzing the
* Corresponding author. No. 1 Tiansheng Road, Beibei, Chongqing 400715, China. Tel.: þ86 23 68253021; fax: þ86 23 68252365. E-mail address: [email protected] (K. Luo). 0885-5765/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.pmpp.2012.11.001
hydrolysis of chitin and b-1,3-glucan, the main cell wall components of many pathogenic fungi [6,7]. Overexpression of chitinase genes from plants or fungi in transformed plants enhances protection against fungal attacks [8e13]. In oilseed rape, transgenic plants that express a hybrid endochitinase gene under a constitutive promoter exhibited an increased resistance to S. sclerotiorum when compared with the non-transgenic parental plants [14]. Recently, overexpression of B. napus MPK4 enhanced resistance to S. sclerotiorum in oilseed rape [15], suggesting that MPK4 positively regulates jasmonic acid-mediated defense response, which might play an important role in resistance to S. sclerotiorum in oilseed rape. A more effective strategy for enhancing resistance to the Sclerotinia pathogen is to degrade oxalic acid (OA), a plant toxin and a key pathogenicity factor secreted by S. sclerotiorum [16,17]. Oxalate oxidase (OXO) can oxidize OA into CO2 and H2O2. Transgenic soybean (Glycine max) and sunflower (Helianthus annuus) that overexpress wheat (Triticum aestivum) OXO have increased resistance to S. sclerotiorum [18e20]. When the same gene was introduced into oilseed rape, a high level of S. sclerotiorum resistance was observed [21]. However, a single antimicrobial gene was introduced into transgenic plants, thus enhanced resistance only
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occurred against a limited number of pathogens [22,23]. The combined expression of multiple antimicrobial proteins was attempted to achieve higher levels of fungal resistance and to broaden the spectrum of resistance against fungal pathogens. Therefore, the isolation of more novel antimicrobial genes from different organisms for use in oilseed rape engineering is necessary to confer resistance against S. sclerotiorum. Recent studies have shown that several antimicrobial proteins (AMPs) play important role in the mechanisms of plant disease resistance [24e26]. AMP expression in transgenic plants enhances resistance to bacterial and fungal pathogens [26e31]. LJAMP2, a heat-stable AMP, was previously isolated from the seeds of motherwort (Leonurus japonicus) [25]. LJAMP2 overexpression significantly improved the resistance and plant-defense responses of tomato (Lycopersicon esculentum L.) and tobacco (Nicotiana tabacum L.) [32,33]. In our previous study, enhanced resistance to fungal pathogens was observed in transgenic Populus tomentosa Carr. overexpressing the LJAMP2 gene [34]. These results indicate that the constitutive expression of the LJAMP2 gene can be exploited to improve resistance to fungal pathogens in plants. In the present study, LJAMP2 was introduced into oilseed rape (Zhongyou 821) using Agrobacterium-mediated transformation, and transgenic oilseed rape plants are evaluated for resistance to S. sclerotiorum infection.
the DNA of putative transgenic plants was performed by polymerase chain reaction (PCR) analysis. Primers were F-nptII (50 CCAACGCTATGTCCTGATAG-30 ) and R-nptII (50 -CTGAATGAACTCCAGGC GAG-30 ) for nptII; F-LJAMP2 (50 -ATGGCTGCCTTGATCAAGTTG-30 ) and R-LJAMP2 (50 -CAGTGCACCTTTGAGCAATC-30 ) for LJAMP2. Each PCR reaction mixture (25 ml) contained 5 mM of each primer, 100 mM of each dNTP, 1 of the supplied Taq buffer, 1.5 mM MgCl2, 2.5 units of Taq DNA polymerase (TakaRa, Dalian, China) and 50 ng of plant genomic DNA as template. PCR conditions were 34 cycles of amplification (94 C for 30 s, 56 C for 30 s, and 72 C for 1 min) in total. DNA samples were separated on a 0.8% (w/v) agarose gel and visualized after ethidium bromide staining.
2. Materials and methods
2.4. In vitro bioassays
2.1. Plant materials, vector and transformation
Lesion sizes were examined by the excised leaf tissue inoculation (ELTI) method [38]. S. sclerotiorum was inoculated on the excised leaves of plants. The cultures were then placed in the glasses containing 50 ml of distilled water to keep the leaves fresh (at 25 2 C), and results were calculated every day after infection. Percentage of spreading lesions was measured by Photoshop CS4 (Adobe) and calculated by statistical analysis (ratios of spreading lesions and the total lesions). For inhibition zone measurement, fresh leaves (5 g) were powdered by friction under liquid nitrogen, and then mixed with 30 ml extraction buffer (50 mM MES, 100 mM TrisCl, 0.1 mM EDTA, 30 mM NaCl, pH 7.0). Ground tissues were then centrifuged at 10,000 g for 10 min at room temperature and the extract collected from each sample. The supernatant is referred to as the crude leaf extract. In vitro antifungal activity assay was performed as described by Jia et al. [13]. Mycelia plug of S. sclerotiorum was inoculated on the center of potato dextrose agar (PDA) medium, which was mixed with 2 ml of crude leaf extracts of each transgenic plants. Inhibition zone was visualized after incubated in the dark for 48 h.
Oilseed rape (Zhongyou 821), kindly provided by Dr. Yourong Chai (Southwest University, China) was used as experimental material. For plant transformation, LJAMP2 was cloned into the pBIN19 vector [35], in which the neomycin phosphotransferase II (NPTII) gene and b-glucuronidase (GUS) gene were plant-selectable marker and the reporter gene, respectively. The resulting vector pBINLJAMP2 was transferred to Agrobacterium strain LBA4404 and single colony was incubated in liquid Murashige and Skoog (MS) medium [36] supplemented with 200 mM acetoneesyringone at 28 C and constant shaking (200 rpm) overnight. Hypocotyl segments (5e10 mm) were cut into from one-weekold seedlings of oilseed rape and pre-cultured in MS medium with 1.0 mg l1 6-BA and 1.0 mg l1 2,4-D for 3 days, then dipped into the diluted Agrobacterium culture for 3e5 min. The hypocotyls were transferred to MS medium with 1.0 mg l1 2,4-D, 1.0 mg l1 6-BA and 200 mM acetoneesyringone. After co-cultivation in the dark for 2 days, the infected hypocotyls were transferred to callus induction medium containing 1.0 mg l1 6-BA, 1.0 mg l1 2,4-D, 500 mg l1 cefotaxime, 100 mg l1 kanamycin and 0.8% (w/v) agar, and maintained at 25 C and 16-h photoperiod of 100 mmol m2 s1. After 2e3 weeks of culture, regenerated calli were subcultured on screening medium (MS medium, 0.8% (w/v) agar, 2.0 mg l1 zeatin, 4.0 mg l1 6-BA, 500 mg l1 cefotaxime and 100 mg l1 kanamycin) to induce shoots. Adventitious shoots were transferred to the medium containing 2.0 mg l1 zeatin, 3.0 mg l1 6-BA, 500 mg l1 cefotaxime and 50 mg l1 kanamycin for growing up, and two weeks later transferred to the rooting medium containing 0.2 mg l1 IBA, 500 mg l1 cefotaxime, 50 mg l1 kanamycin. Rooted plantlets were acclimatized in pots at 25 C in a 16-h photoperiod, finally grew them to the greenhouse for further studies. 2.2. DNA extraction and PCR analysis Total genomic DNA was extracted from leaf of wild-type and transformed plants via modified cetyltrimethyl-ammonium bromide (CTAB) method [13]. To detect the presence of the transgene,
2.3. Histochemical staining for GUS activity Histochemical staining for b-glucuronidase (GUS) activity was performed according to the method of Jefferson et al. [37]. The leaves of 4-week-old transgenic oilseed rape plants were incubated in the GUS reaction buffer (1 mM 5-bromo-4-chloro-3-indolyl-b-Dglucuronide, 100 mM phosphate buffer, pH 7.0, 5 mM K3[Fe(CN)6], 5 mM K4[Fe(CN)6], and 10 mM EDTA) at 37 C for 18 h. For visualization of the stained tissue, leaves were rinsed with an ethanol series (100%, 75% and 50%) to remove chlorophyll at room temperature, and then mounted for photographing.
2.5. Mycelia staining with trypan blue Growth of S. sclerotiorum on the transgenic plants and the untransformed control was examined by trypan blue staining according to the method of Wang et al. [15]. In brief, leaves of plants were inoculated with S. sclerotiorum and soaked with 100% ethanol for 3 h, then placed in trypan blue solution for 4 h and incubated in chloral hydrate for 24 h. Growth of mycelium was viewed with a fluorescent microscope (Nikon eclipse 80i). 2.6. RNA extraction and RT-PCR analysis Total RNA was extracted from wild-type and transgenic oilseed rape plants using RNA RNeasy Plant Mini Kit (Qiagen, Germany). DNase-treated RNA (2.0 g) was reverse-transcribed in a total volume of 20 l using RT-AMV transcriptase (TaKaRa, Dalian, China) with oligo (dT) at 42 C for 30 min followed by 24 cycles of amplification (94 C for 1 min, 56 C for 30 s, and 72 C for 1 min),
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finally 7 min at 72 C. Individual RT-PCR products were resolved by agarose gel electrophoresis and visualized with ethidium bromide under UV light. The following primers: F-PDF1.2 (50 -CATCACCCTTCTCTTCGCTGC-30 ) and R-PDF1.2 (50 -ATGTCCCACTTGACC TCTCGC-30 ) were used to checking PDF1.2 gene (AY884023) as well as primers: F-PR-1 (50 -TGCCAACGCTCACAACCA-30 ) and R-PR-1 (50 ACGGGACCTACGCC TACT-30 ) for PR-1 gene (AY623008). 2.7. DAB staining The accumulation of H2O2 in leaves of transgenic plants was observed by 30 ,3-diaminobenzidine (DAB) staining [39]. Leaves were sprayed with salicylic acid (SA) solution (5 mM, pH 5.8) and then placed in a plastic box at 25 C. After 10 h, leaves were soaked with DAB solution (1 mg ml1, pH 3.8) for 12 h and then incubated in 100% ethanol for 4 h to destain. 3. Results 3.1. Generation of transgenic oilseed rape plants The pBIN19 vector containing the LJAMP2 gene from motherwort [35] was used in the transformation of oilseed rape (Fig. 1A). Infected explants were cultured on a selective medium supplemented with 100 mg l1 kanamycin for selecting positive calli. The explants with positive calli were then subcultured on a shoot induction medium, and a number of adventitious shoots were regenerated from these explants about 8 weeks after infection. The kanamycin-resistant putative transformants were rooted and then grown in a greenhouse. The growth and development of putative transgenic lines were similar to wild-type plants, and no abnormal phenotypes were observed (data not shown), suggesting that the constitutive expression of LJAMP2 did not cause detectable morphologic alterations in oilseed rape plants.
Fig. 1. Transformation of oilseed rape (Brassica napus) with LJAMP2. A, the T-DNA region of plant expression vector pBIN-LJAMP2. RB, right border; 35S, cauliflower mosaic virus 35S promoter; Nos, Nos terminator; NPTII, neomycin phosphotransferase II gene; GUS, b-glucuronidase gene; LB, left border. B, PCR analysis of LJAMP2 transgenic plants with primers specific to LJAMP2 and NPTII genes. WT, the untransformed control; lanes #2-1 to #8-2, transgenic plants; þ, positive control (pBIN-LJAMP2); , negative control (H2O). C, histochemical analysis of GUS activity in leaves of wild-type and transgenic oilseed rape plants. Left, transgenic plants (#2-1); right, the wild-type control.
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The putative T0 transformants were confirmed through GUS histochemical staining [37]. GUS activity was detected in the leaves of these transformed plants with kanamycin resistance, but was absent in untransformed plants (Fig. 1C). The independent GUSpositive transgenic lines were further verified by PCR analysis using gene-specific primers for NPTII and LJAMP2, respectively. The expected 500-bp and 375-bp PCR products, representing the NPTII gene and the fragment specific to the LJAMP2 gene, respectively, were observed in all GUS-positive transgenic lines (Fig. 1B). No amplification signal was detected in untransformed plants. These results indicated that the NPTII and LJAMP2 genes had been stably integrated into the genome of the tested transgenic oilseed rape plants. 3.2. Transgenic plants enhance resistance to S. sclerotiorum To determine the antifungal activity of the motherwort LJAMP2 protein in vitro, crude leaf extracts from transgenic oilseed rape plants were obtained and tested for activity against S. sclerotiorum. The mycelial growth of the pathogen was significantly inhibited by the crude extracts from transgenic LJAMP2 lines, whereas no inhibition zone was observed with the supplement of the extracts from the untransformed plants (Fig. 2), suggesting that LJAMP2 overexpression could lead to enhanced resistance against the fungal pathogen S. sclerotiorum in transgenic oilseed rape plants. To further evaluate the disease resistance of transgenic oilseed rape overexpressing LJAMP2 against the fungal pathogen S. sclerotiorum, a standard leaf assay was performed as described by Jia et al. [34]. The hyphae of S. sclerotiorum were inoculated onto the surface of excised leaves of three individual transgenic lines and untransformed plants. Typical disease symptoms were observed in the wild-type control plants approximately 3 days after infection, and larger necrotic lesions appeared on the infected leaves 4 days later which resulted in extensive tissue damage (Fig. 3A). In contrast, all transgenic LJAMP2 lines exhibited a delayed in the appearance of necrotic lesions after infection. The lesion sizes in transgenic lines #2-1, #6-5, and #8-1 were reduced by 14%, 46%, and 38%, respectively, compared with that of the control plants (Fig. 3B). The relatively lower resistance to S. sclerotiorum was found in transgenic line #2-1, but no significant differences were observed between these transgenic lines tested. To investigate the expression of the transgene in transgenic oilseed rape plants at transcriptional level, RT-PCR analysis was performed using gene-specific primers for LJAMP2. Total RNA was extracted from the leaves of three selected transgenic lines and wild-type plants. The results showed that LJAMP2 expression was detected in all transgenic lines, whereas no accumulation of LJAMP2 mRNA was found in the untransformed plants (Fig. 3C).
Fig. 2. In vitro antifungal activity of LJAMP2. PDA culture medium was contained with crude leaf extracts of transgenic plant and wild-type. Fungal mycelia plug was placed in the center of plate. Disease mycelium sizes were shown at 48 h after inoculation. The experiments were repeated three times. Left, transgenic plants (#6-3); right, the wild-type control.
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Fig. 3. Resistance of LJAMP2 transgenic plants to S. sclerotiorum. A, excised leaf assays for resistance to S. sclerotiorum in transgenic lines #2-1, #6-5, #8-1 and the untransformed wild-type (WT) control. These detached leaves were inoculated with the fungal mycelia plugs of S. sclerotiorum and covered with a piece of plastic wrap and then incubated in a greenhouse at 25 2 C. The experiments were repeated three times. B, disease lesion sizes were determined from 1 to 7 days after inoculation. The error bars indicate standard deviation from three replicates. C, expression levels of transgenic lines was estimated with LJAMP2-specific primers by semi-quantitative RT-PCR.
The growth and development of S. sclerotiorum mycelia inside the leaves of the wild-type and transgenic plants after infection were determined using trypan blue staining. In contrast to the untransformed control plants, the transgenic plants that expressed LJAMP2 inhibited mycelial growth of S. sclerotiorum within 15 h after inoculation (Fig. 4). Mycelial growth in the untransformed control leaves appeared normal, whereas mycelia became curly and relatively shorter and thicker in the transgenic plants. These data, consistent with the results above, clearly indicate that LJAMP2 overexpression in oilseed rape inhibited the mycelial growth of S. sclerotiorum, resulting in an increased resistance to fungal pathogens. 3.3. Expression analysis of defense-related genes in transgenic LJAMP2 plants To characterize whether LJAMP2 overexpression resulted in expression changes in the defense-marker genes of transgenic plants, a semi-quantitative RT-PCR was used to detect the time course of the PDF1.2 and PR-1 transcript levels as an indicator of resistance. PDF1.2 and PR-1 are considered marker genes in the JAand SA-related defense pathways, respectively [40e42]. PR-1 expression was at a low level in uninoculated transgenic plants, but was strongly induced 12 h after infection by S. sclerotiorum (Fig. 5A), indicating that LJAMP2 overexpression in oilseed rape led to the activation of the plant defense-related gene PR-1 upon fungal pathogen induction. In contrast, PDF1.2 expression was not induced in the transgenic LJAMP2 plants compared with the wild-type plants (Fig. 5B). Therefore, the infection might have activated the SA-related defense responses, but had no effect on the JA-related defense responses in the transgenic LJAMP2 oilseed rape. 3.4. LJAMP2 expression induces H2O2 accumulation in transgenic plants H2O2 is an important mediator that contributes to defense response during plantepathogen interactions, and is a key factor
Fig. 4. Inhibition of S. sclerotiorum mycelial growth by overexpression of LJAMP2 in Brassica napus. Mycelial growth was determined by using trypan blue staining 5, 10, 15 and 20 h after inoculation on leaves of the wild-type and transgenic plants. Details of mycelial growth at 10 h were observed. Left, transgenic plants (#6-5); right, control (wild-type). The experiments were repeated twice with transgenic line #8-1 and the same results were obtained.
that regulates programmed cell death in pathogen, evocator, and hormone responses [43]. To determine whether LJAMP2 overexpression resulted in changes in H2O2 accumulation, transgenic and untransformed plants were analyzed using 30 ,3diaminobenzidine (DAB) staining. The transgenic LJAMP2 plants produced less redebrown precipitate in their leaves compared with the untransformed control (Fig. 6), indicating that higher levels of H2O2 were accumulated in transgenic oil rape plants. 4. Discussion The pathogen S. sclerotiorum is a major fungal plantepathogen that attacks over 400 species of host plants [5,44] and causes significant yield losses in several important crops worldwide [45]. Transgenic plants that resist S. sclerotiorum have been reported in soybean [18,46], sunflower [47], lettuce [48], and peanuts [49]. In oilseed rape, Dong et al. [21] showed that transgenic oilseed rape overexpressing wheat oxalate oxidase had enhanced resistance to S. sclerotiorum. More recently, MPK4 overexpression, which positively regulates jasmonic acid-mediated response, has been demonstrated to improve resistance to S. sclerotiorum in oilseed rape [15]. In the present paper, we reported the overexpression of an antimicrobial protein LJAMP2 from motherwort (L. japonicus Houtt) in oilseed rape. In vitro experiments revealed that the mycelial growth of S. sclerotiorum was significantly inhibited when supplied with crude leaf extracts from transgenic LJAMP2 plants (Fig. 2), indicating that LJAMP2 protein possesses antifungal activity. This result is consistent with previous studies [25,34]. Further studies showed that transgenic oilseed rape plants that overexpress LJAMP2 exhibited enhanced resistance to the fungal pathogen S. sclerotiorum (Fig. 3A and B). Therefore, the constitutive
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Fig. 5. Responses of PR-1 and PDF1.2 genes by overexpression of LJAMP2 in oilseed rape plants. Relative expression levels of PR-1 and PDF1.2 in transgenic plants were determined by semi-quantitative RT-PCR at 0, 6, and 12 h after inoculation of S. sclerotiorum. WT was the untransformed wild-type plants.
expression of the LJAMP2 gene can be used to increase the resistance of oilseed rape to S. sclerotiorum. LJAMP2 belongs to the plant non-specific lipid transfer protein (nsLTP) family, whose role in plant defense is mainly related to antimicrobial activity [25,50]. Increasing evidence has shown that nsLTPs play an important role in plant defense against viral, bacterial, and fungal pathogens [34,35,50e53]. For instance, Ace-AMP1 overexpression in scented geranium confers resistance to the necrotrophic fungi Botrytis cinerea [54]. Pn-AMPs cause decreased susceptibility to phytopathogenic fungi in transgenic tomatoes [55]. Previous studies have demonstrated that LJAMP2 overexpression in tobacco and poplar plants significantly enhances resistance to bacterial and fungal pathogens, such as Ralstonia solanacearum, Alternaria alternata, Alternaria alternata (Fr.) Keissler, and Colletotrichum gloeosporioides (Penz.) [34,35]. Our results showed that the transgenic LJAMP2 oilseed rape also exhibited resistance to S. sclerotiorum, suggesting that LJAMP2 has a potential application in improving disease resistance in crop plants. SA and JA play important roles in the signal transduction of plant-defense systems. In general, SA is implicated in disease resistance against biotrophic pathogens, whereas JA participates in the response to necrotrophs [56]. Zhao et al. [57] and Mao et al. [58] suggested that JA- and ET-dependent pathways were induced by S. sclerotiorum infection in oilseed rape. However, a study based on the Arabidopsis mutant suggested that, in addition to the JA/ET-
mediated defense response, the SA-mediated defense response is also involved in defense against S. sclerotiorum [59]. In our current study, the expression level of PR-1 was elevated, whereas PDF1.2 transcript levels did not increase in the LJAMP2 lines when infected with S. sclerotiorum (Fig. 5), suggesting that LJAMP2 overexpression induced by S. sclerotiorum might be mediated by the SA-dependent pathway. Thus, it is an oversimplification to conclude strict separation of defenses against necrotrophic and biotrophic pathogens via SA and JA/ET signaling, respectively, based on these studies on S. sclerotiorum [60,61]. SA has a close relationship with H2O2 in plant disease resistance. Several studies have shown that SA may be related to the production of H2O2 [62e64]. The role of SA in plants may be to increase the H2O2 content through catalase inhibition [65]. The exogenous application of SA in the transgenic lines induced a significantly greater accumulation of H2O2 compared with the non-transformed plants (Fig. 6). This is consistent with our results in which the SAmediated signaling pathway was enhanced in LJAMP2 transgenic plants. Therefore, the overexpression of the LJAMP2 gene in oilseed rape in our study may provide new data on the relationship between AMPs and the disease resistance signaling pathway of the plant-defense system. Acknowledgments The authors thank Dr. Yourong Cai (Southwest University, Chongqing, China) for providing fungal stain S. sclerotiorum. This work was supported by the National Natural Science Foundation of China (31171620), the National Key Project for Research on Transgenic Plant (2011ZX08010-003), the program for New Century Excellent Talents in University (NCET-11-0700) and The Research Fund for the Doctoral Program of Higher Education (20110182110004). References
Fig. 6. H2O2 accumulation in oilseed rape plants overexpressing LJAMP2. In situ detection of H2O2 was performed by using 3, 3-diaminobenzidine staining in the untransformed wild-type (WT) control and transgenic line (#6-5).
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Physiological and Molecular Plant Pathology journal homepage: www.elsevier.com/locate/pmpp
Overexpression of an nsLTPs-like antimicrobial protein gene (LJAMP2) from motherwort (Leonurus japonicus) enhances resistance to Sclerotinia sclerotiorum in oilseed rape (Brassica napus) Yuanzhong Jiang a, Xialan Fu a, Mengling Wen a, Fei Wang a, Qiao Tang a, Qiaoyan Tian a, Keming Luo a, b, * a
State Key Laboratory of Eco-Environment and Bio-Resource of Three Gorges Reservoir Region, Key Laboratory of Eco-Environments of Three Gorges Reservoir Region, Ministry of Education, Institute of Resources Botany, School of Life Sciences, Southwest University, Chongqing 400715, China Key Laboratory of Biotechnology and Crop Quality Improvement of Ministry of Agriculture of China, Biotechnology Research Center, Southwest University, Chongqing 400715, China
b
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 3 November 2012
Sclerotinia stem rot caused by Sclerotinia sclerotiorum is one of the most important diseases of oilseed rape worldwide and leads to considerable yield losses. In this study, a non-specific lipid transfer proteinlike antimicrobial protein gene (LJAMP2) from motherwort (Leonurus japonicus) was introduced into oilseed rape (Zhongyou 821) by Agrobacterium-mediated transformation. In vitro experiments revealed that the mycelial growth of S. sclerotiorum was significantly inhibited when supplied with crude leaf extracts from transgenic oilseed rape plants overexpressing LJAMP2. Furthermore, in vivo studies showed that transgenic LJAMP2 plants had enhanced resistance to S. sclerotiorum. Semi-quantitative RT-PCR analysis showed that the LJAMP2 gene was transcribed in all transformed plants. In addition, we also found that overexpression of LJAMP2 in transgenic plants caused constitutive activation of the defenserelated gene PR-1 and an increase of H2O2 production, but did not enhance PDF1.2 expression. Our results suggest that constitutive expression of the LJAMP2 gene from motherwort seeds might be exploited to improve the resistance of oilseed rape against S. sclerotiorum. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Sclerotinia sclerotiorum Antimicrobial protein Oilseed rape LJAMP2
1. Introduction Oilseed rape (Brassica napus L.) is the third largest oilseed crop in the world and provides approximately 13% of the world’s supply of vegetable oil [1]. However, oilseed rape can be seriously infected by Sclerotinia sclerotiorum, which causes the rotting of stems and leaves, resulting in tremendous yield loss worldwide [2]. Due to the lack of endogenous resistance resources in cruciferous plants, little progress has been achieved in the breeding of Sclerotinia resistance in oilseed rape by applying traditional breeding methods [3]. Chemical control of S. sclerotiorum is expensive, often ineffective, and causes environmental pollution. Therefore, the transgenic modification of the crop shows promise for the production of new oilseed rape lines with greater resistance to S. sclerotiorum. S. sclerotiorum, as a necrotrophic fungal pathogen that occurs worldwide, is pathogenic to more than 400 plant species [4,5]. Chitinase and b-1,3-glucanase are capable of catalyzing the
* Corresponding author. No. 1 Tiansheng Road, Beibei, Chongqing 400715, China. Tel.: þ86 23 68253021; fax: þ86 23 68252365. E-mail address: [email protected] (K. Luo). 0885-5765/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.pmpp.2012.11.001
hydrolysis of chitin and b-1,3-glucan, the main cell wall components of many pathogenic fungi [6,7]. Overexpression of chitinase genes from plants or fungi in transformed plants enhances protection against fungal attacks [8e13]. In oilseed rape, transgenic plants that express a hybrid endochitinase gene under a constitutive promoter exhibited an increased resistance to S. sclerotiorum when compared with the non-transgenic parental plants [14]. Recently, overexpression of B. napus MPK4 enhanced resistance to S. sclerotiorum in oilseed rape [15], suggesting that MPK4 positively regulates jasmonic acid-mediated defense response, which might play an important role in resistance to S. sclerotiorum in oilseed rape. A more effective strategy for enhancing resistance to the Sclerotinia pathogen is to degrade oxalic acid (OA), a plant toxin and a key pathogenicity factor secreted by S. sclerotiorum [16,17]. Oxalate oxidase (OXO) can oxidize OA into CO2 and H2O2. Transgenic soybean (Glycine max) and sunflower (Helianthus annuus) that overexpress wheat (Triticum aestivum) OXO have increased resistance to S. sclerotiorum [18e20]. When the same gene was introduced into oilseed rape, a high level of S. sclerotiorum resistance was observed [21]. However, a single antimicrobial gene was introduced into transgenic plants, thus enhanced resistance only
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occurred against a limited number of pathogens [22,23]. The combined expression of multiple antimicrobial proteins was attempted to achieve higher levels of fungal resistance and to broaden the spectrum of resistance against fungal pathogens. Therefore, the isolation of more novel antimicrobial genes from different organisms for use in oilseed rape engineering is necessary to confer resistance against S. sclerotiorum. Recent studies have shown that several antimicrobial proteins (AMPs) play important role in the mechanisms of plant disease resistance [24e26]. AMP expression in transgenic plants enhances resistance to bacterial and fungal pathogens [26e31]. LJAMP2, a heat-stable AMP, was previously isolated from the seeds of motherwort (Leonurus japonicus) [25]. LJAMP2 overexpression significantly improved the resistance and plant-defense responses of tomato (Lycopersicon esculentum L.) and tobacco (Nicotiana tabacum L.) [32,33]. In our previous study, enhanced resistance to fungal pathogens was observed in transgenic Populus tomentosa Carr. overexpressing the LJAMP2 gene [34]. These results indicate that the constitutive expression of the LJAMP2 gene can be exploited to improve resistance to fungal pathogens in plants. In the present study, LJAMP2 was introduced into oilseed rape (Zhongyou 821) using Agrobacterium-mediated transformation, and transgenic oilseed rape plants are evaluated for resistance to S. sclerotiorum infection.
the DNA of putative transgenic plants was performed by polymerase chain reaction (PCR) analysis. Primers were F-nptII (50 CCAACGCTATGTCCTGATAG-30 ) and R-nptII (50 -CTGAATGAACTCCAGGC GAG-30 ) for nptII; F-LJAMP2 (50 -ATGGCTGCCTTGATCAAGTTG-30 ) and R-LJAMP2 (50 -CAGTGCACCTTTGAGCAATC-30 ) for LJAMP2. Each PCR reaction mixture (25 ml) contained 5 mM of each primer, 100 mM of each dNTP, 1 of the supplied Taq buffer, 1.5 mM MgCl2, 2.5 units of Taq DNA polymerase (TakaRa, Dalian, China) and 50 ng of plant genomic DNA as template. PCR conditions were 34 cycles of amplification (94 C for 30 s, 56 C for 30 s, and 72 C for 1 min) in total. DNA samples were separated on a 0.8% (w/v) agarose gel and visualized after ethidium bromide staining.
2. Materials and methods
2.4. In vitro bioassays
2.1. Plant materials, vector and transformation
Lesion sizes were examined by the excised leaf tissue inoculation (ELTI) method [38]. S. sclerotiorum was inoculated on the excised leaves of plants. The cultures were then placed in the glasses containing 50 ml of distilled water to keep the leaves fresh (at 25 2 C), and results were calculated every day after infection. Percentage of spreading lesions was measured by Photoshop CS4 (Adobe) and calculated by statistical analysis (ratios of spreading lesions and the total lesions). For inhibition zone measurement, fresh leaves (5 g) were powdered by friction under liquid nitrogen, and then mixed with 30 ml extraction buffer (50 mM MES, 100 mM TrisCl, 0.1 mM EDTA, 30 mM NaCl, pH 7.0). Ground tissues were then centrifuged at 10,000 g for 10 min at room temperature and the extract collected from each sample. The supernatant is referred to as the crude leaf extract. In vitro antifungal activity assay was performed as described by Jia et al. [13]. Mycelia plug of S. sclerotiorum was inoculated on the center of potato dextrose agar (PDA) medium, which was mixed with 2 ml of crude leaf extracts of each transgenic plants. Inhibition zone was visualized after incubated in the dark for 48 h.
Oilseed rape (Zhongyou 821), kindly provided by Dr. Yourong Chai (Southwest University, China) was used as experimental material. For plant transformation, LJAMP2 was cloned into the pBIN19 vector [35], in which the neomycin phosphotransferase II (NPTII) gene and b-glucuronidase (GUS) gene were plant-selectable marker and the reporter gene, respectively. The resulting vector pBINLJAMP2 was transferred to Agrobacterium strain LBA4404 and single colony was incubated in liquid Murashige and Skoog (MS) medium [36] supplemented with 200 mM acetoneesyringone at 28 C and constant shaking (200 rpm) overnight. Hypocotyl segments (5e10 mm) were cut into from one-weekold seedlings of oilseed rape and pre-cultured in MS medium with 1.0 mg l1 6-BA and 1.0 mg l1 2,4-D for 3 days, then dipped into the diluted Agrobacterium culture for 3e5 min. The hypocotyls were transferred to MS medium with 1.0 mg l1 2,4-D, 1.0 mg l1 6-BA and 200 mM acetoneesyringone. After co-cultivation in the dark for 2 days, the infected hypocotyls were transferred to callus induction medium containing 1.0 mg l1 6-BA, 1.0 mg l1 2,4-D, 500 mg l1 cefotaxime, 100 mg l1 kanamycin and 0.8% (w/v) agar, and maintained at 25 C and 16-h photoperiod of 100 mmol m2 s1. After 2e3 weeks of culture, regenerated calli were subcultured on screening medium (MS medium, 0.8% (w/v) agar, 2.0 mg l1 zeatin, 4.0 mg l1 6-BA, 500 mg l1 cefotaxime and 100 mg l1 kanamycin) to induce shoots. Adventitious shoots were transferred to the medium containing 2.0 mg l1 zeatin, 3.0 mg l1 6-BA, 500 mg l1 cefotaxime and 50 mg l1 kanamycin for growing up, and two weeks later transferred to the rooting medium containing 0.2 mg l1 IBA, 500 mg l1 cefotaxime, 50 mg l1 kanamycin. Rooted plantlets were acclimatized in pots at 25 C in a 16-h photoperiod, finally grew them to the greenhouse for further studies. 2.2. DNA extraction and PCR analysis Total genomic DNA was extracted from leaf of wild-type and transformed plants via modified cetyltrimethyl-ammonium bromide (CTAB) method [13]. To detect the presence of the transgene,
2.3. Histochemical staining for GUS activity Histochemical staining for b-glucuronidase (GUS) activity was performed according to the method of Jefferson et al. [37]. The leaves of 4-week-old transgenic oilseed rape plants were incubated in the GUS reaction buffer (1 mM 5-bromo-4-chloro-3-indolyl-b-Dglucuronide, 100 mM phosphate buffer, pH 7.0, 5 mM K3[Fe(CN)6], 5 mM K4[Fe(CN)6], and 10 mM EDTA) at 37 C for 18 h. For visualization of the stained tissue, leaves were rinsed with an ethanol series (100%, 75% and 50%) to remove chlorophyll at room temperature, and then mounted for photographing.
2.5. Mycelia staining with trypan blue Growth of S. sclerotiorum on the transgenic plants and the untransformed control was examined by trypan blue staining according to the method of Wang et al. [15]. In brief, leaves of plants were inoculated with S. sclerotiorum and soaked with 100% ethanol for 3 h, then placed in trypan blue solution for 4 h and incubated in chloral hydrate for 24 h. Growth of mycelium was viewed with a fluorescent microscope (Nikon eclipse 80i). 2.6. RNA extraction and RT-PCR analysis Total RNA was extracted from wild-type and transgenic oilseed rape plants using RNA RNeasy Plant Mini Kit (Qiagen, Germany). DNase-treated RNA (2.0 g) was reverse-transcribed in a total volume of 20 l using RT-AMV transcriptase (TaKaRa, Dalian, China) with oligo (dT) at 42 C for 30 min followed by 24 cycles of amplification (94 C for 1 min, 56 C for 30 s, and 72 C for 1 min),
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finally 7 min at 72 C. Individual RT-PCR products were resolved by agarose gel electrophoresis and visualized with ethidium bromide under UV light. The following primers: F-PDF1.2 (50 -CATCACCCTTCTCTTCGCTGC-30 ) and R-PDF1.2 (50 -ATGTCCCACTTGACC TCTCGC-30 ) were used to checking PDF1.2 gene (AY884023) as well as primers: F-PR-1 (50 -TGCCAACGCTCACAACCA-30 ) and R-PR-1 (50 ACGGGACCTACGCC TACT-30 ) for PR-1 gene (AY623008). 2.7. DAB staining The accumulation of H2O2 in leaves of transgenic plants was observed by 30 ,3-diaminobenzidine (DAB) staining [39]. Leaves were sprayed with salicylic acid (SA) solution (5 mM, pH 5.8) and then placed in a plastic box at 25 C. After 10 h, leaves were soaked with DAB solution (1 mg ml1, pH 3.8) for 12 h and then incubated in 100% ethanol for 4 h to destain. 3. Results 3.1. Generation of transgenic oilseed rape plants The pBIN19 vector containing the LJAMP2 gene from motherwort [35] was used in the transformation of oilseed rape (Fig. 1A). Infected explants were cultured on a selective medium supplemented with 100 mg l1 kanamycin for selecting positive calli. The explants with positive calli were then subcultured on a shoot induction medium, and a number of adventitious shoots were regenerated from these explants about 8 weeks after infection. The kanamycin-resistant putative transformants were rooted and then grown in a greenhouse. The growth and development of putative transgenic lines were similar to wild-type plants, and no abnormal phenotypes were observed (data not shown), suggesting that the constitutive expression of LJAMP2 did not cause detectable morphologic alterations in oilseed rape plants.
Fig. 1. Transformation of oilseed rape (Brassica napus) with LJAMP2. A, the T-DNA region of plant expression vector pBIN-LJAMP2. RB, right border; 35S, cauliflower mosaic virus 35S promoter; Nos, Nos terminator; NPTII, neomycin phosphotransferase II gene; GUS, b-glucuronidase gene; LB, left border. B, PCR analysis of LJAMP2 transgenic plants with primers specific to LJAMP2 and NPTII genes. WT, the untransformed control; lanes #2-1 to #8-2, transgenic plants; þ, positive control (pBIN-LJAMP2); , negative control (H2O). C, histochemical analysis of GUS activity in leaves of wild-type and transgenic oilseed rape plants. Left, transgenic plants (#2-1); right, the wild-type control.
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The putative T0 transformants were confirmed through GUS histochemical staining [37]. GUS activity was detected in the leaves of these transformed plants with kanamycin resistance, but was absent in untransformed plants (Fig. 1C). The independent GUSpositive transgenic lines were further verified by PCR analysis using gene-specific primers for NPTII and LJAMP2, respectively. The expected 500-bp and 375-bp PCR products, representing the NPTII gene and the fragment specific to the LJAMP2 gene, respectively, were observed in all GUS-positive transgenic lines (Fig. 1B). No amplification signal was detected in untransformed plants. These results indicated that the NPTII and LJAMP2 genes had been stably integrated into the genome of the tested transgenic oilseed rape plants. 3.2. Transgenic plants enhance resistance to S. sclerotiorum To determine the antifungal activity of the motherwort LJAMP2 protein in vitro, crude leaf extracts from transgenic oilseed rape plants were obtained and tested for activity against S. sclerotiorum. The mycelial growth of the pathogen was significantly inhibited by the crude extracts from transgenic LJAMP2 lines, whereas no inhibition zone was observed with the supplement of the extracts from the untransformed plants (Fig. 2), suggesting that LJAMP2 overexpression could lead to enhanced resistance against the fungal pathogen S. sclerotiorum in transgenic oilseed rape plants. To further evaluate the disease resistance of transgenic oilseed rape overexpressing LJAMP2 against the fungal pathogen S. sclerotiorum, a standard leaf assay was performed as described by Jia et al. [34]. The hyphae of S. sclerotiorum were inoculated onto the surface of excised leaves of three individual transgenic lines and untransformed plants. Typical disease symptoms were observed in the wild-type control plants approximately 3 days after infection, and larger necrotic lesions appeared on the infected leaves 4 days later which resulted in extensive tissue damage (Fig. 3A). In contrast, all transgenic LJAMP2 lines exhibited a delayed in the appearance of necrotic lesions after infection. The lesion sizes in transgenic lines #2-1, #6-5, and #8-1 were reduced by 14%, 46%, and 38%, respectively, compared with that of the control plants (Fig. 3B). The relatively lower resistance to S. sclerotiorum was found in transgenic line #2-1, but no significant differences were observed between these transgenic lines tested. To investigate the expression of the transgene in transgenic oilseed rape plants at transcriptional level, RT-PCR analysis was performed using gene-specific primers for LJAMP2. Total RNA was extracted from the leaves of three selected transgenic lines and wild-type plants. The results showed that LJAMP2 expression was detected in all transgenic lines, whereas no accumulation of LJAMP2 mRNA was found in the untransformed plants (Fig. 3C).
Fig. 2. In vitro antifungal activity of LJAMP2. PDA culture medium was contained with crude leaf extracts of transgenic plant and wild-type. Fungal mycelia plug was placed in the center of plate. Disease mycelium sizes were shown at 48 h after inoculation. The experiments were repeated three times. Left, transgenic plants (#6-3); right, the wild-type control.
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Fig. 3. Resistance of LJAMP2 transgenic plants to S. sclerotiorum. A, excised leaf assays for resistance to S. sclerotiorum in transgenic lines #2-1, #6-5, #8-1 and the untransformed wild-type (WT) control. These detached leaves were inoculated with the fungal mycelia plugs of S. sclerotiorum and covered with a piece of plastic wrap and then incubated in a greenhouse at 25 2 C. The experiments were repeated three times. B, disease lesion sizes were determined from 1 to 7 days after inoculation. The error bars indicate standard deviation from three replicates. C, expression levels of transgenic lines was estimated with LJAMP2-specific primers by semi-quantitative RT-PCR.
The growth and development of S. sclerotiorum mycelia inside the leaves of the wild-type and transgenic plants after infection were determined using trypan blue staining. In contrast to the untransformed control plants, the transgenic plants that expressed LJAMP2 inhibited mycelial growth of S. sclerotiorum within 15 h after inoculation (Fig. 4). Mycelial growth in the untransformed control leaves appeared normal, whereas mycelia became curly and relatively shorter and thicker in the transgenic plants. These data, consistent with the results above, clearly indicate that LJAMP2 overexpression in oilseed rape inhibited the mycelial growth of S. sclerotiorum, resulting in an increased resistance to fungal pathogens. 3.3. Expression analysis of defense-related genes in transgenic LJAMP2 plants To characterize whether LJAMP2 overexpression resulted in expression changes in the defense-marker genes of transgenic plants, a semi-quantitative RT-PCR was used to detect the time course of the PDF1.2 and PR-1 transcript levels as an indicator of resistance. PDF1.2 and PR-1 are considered marker genes in the JAand SA-related defense pathways, respectively [40e42]. PR-1 expression was at a low level in uninoculated transgenic plants, but was strongly induced 12 h after infection by S. sclerotiorum (Fig. 5A), indicating that LJAMP2 overexpression in oilseed rape led to the activation of the plant defense-related gene PR-1 upon fungal pathogen induction. In contrast, PDF1.2 expression was not induced in the transgenic LJAMP2 plants compared with the wild-type plants (Fig. 5B). Therefore, the infection might have activated the SA-related defense responses, but had no effect on the JA-related defense responses in the transgenic LJAMP2 oilseed rape. 3.4. LJAMP2 expression induces H2O2 accumulation in transgenic plants H2O2 is an important mediator that contributes to defense response during plantepathogen interactions, and is a key factor
Fig. 4. Inhibition of S. sclerotiorum mycelial growth by overexpression of LJAMP2 in Brassica napus. Mycelial growth was determined by using trypan blue staining 5, 10, 15 and 20 h after inoculation on leaves of the wild-type and transgenic plants. Details of mycelial growth at 10 h were observed. Left, transgenic plants (#6-5); right, control (wild-type). The experiments were repeated twice with transgenic line #8-1 and the same results were obtained.
that regulates programmed cell death in pathogen, evocator, and hormone responses [43]. To determine whether LJAMP2 overexpression resulted in changes in H2O2 accumulation, transgenic and untransformed plants were analyzed using 30 ,3diaminobenzidine (DAB) staining. The transgenic LJAMP2 plants produced less redebrown precipitate in their leaves compared with the untransformed control (Fig. 6), indicating that higher levels of H2O2 were accumulated in transgenic oil rape plants. 4. Discussion The pathogen S. sclerotiorum is a major fungal plantepathogen that attacks over 400 species of host plants [5,44] and causes significant yield losses in several important crops worldwide [45]. Transgenic plants that resist S. sclerotiorum have been reported in soybean [18,46], sunflower [47], lettuce [48], and peanuts [49]. In oilseed rape, Dong et al. [21] showed that transgenic oilseed rape overexpressing wheat oxalate oxidase had enhanced resistance to S. sclerotiorum. More recently, MPK4 overexpression, which positively regulates jasmonic acid-mediated response, has been demonstrated to improve resistance to S. sclerotiorum in oilseed rape [15]. In the present paper, we reported the overexpression of an antimicrobial protein LJAMP2 from motherwort (L. japonicus Houtt) in oilseed rape. In vitro experiments revealed that the mycelial growth of S. sclerotiorum was significantly inhibited when supplied with crude leaf extracts from transgenic LJAMP2 plants (Fig. 2), indicating that LJAMP2 protein possesses antifungal activity. This result is consistent with previous studies [25,34]. Further studies showed that transgenic oilseed rape plants that overexpress LJAMP2 exhibited enhanced resistance to the fungal pathogen S. sclerotiorum (Fig. 3A and B). Therefore, the constitutive
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Fig. 5. Responses of PR-1 and PDF1.2 genes by overexpression of LJAMP2 in oilseed rape plants. Relative expression levels of PR-1 and PDF1.2 in transgenic plants were determined by semi-quantitative RT-PCR at 0, 6, and 12 h after inoculation of S. sclerotiorum. WT was the untransformed wild-type plants.
expression of the LJAMP2 gene can be used to increase the resistance of oilseed rape to S. sclerotiorum. LJAMP2 belongs to the plant non-specific lipid transfer protein (nsLTP) family, whose role in plant defense is mainly related to antimicrobial activity [25,50]. Increasing evidence has shown that nsLTPs play an important role in plant defense against viral, bacterial, and fungal pathogens [34,35,50e53]. For instance, Ace-AMP1 overexpression in scented geranium confers resistance to the necrotrophic fungi Botrytis cinerea [54]. Pn-AMPs cause decreased susceptibility to phytopathogenic fungi in transgenic tomatoes [55]. Previous studies have demonstrated that LJAMP2 overexpression in tobacco and poplar plants significantly enhances resistance to bacterial and fungal pathogens, such as Ralstonia solanacearum, Alternaria alternata, Alternaria alternata (Fr.) Keissler, and Colletotrichum gloeosporioides (Penz.) [34,35]. Our results showed that the transgenic LJAMP2 oilseed rape also exhibited resistance to S. sclerotiorum, suggesting that LJAMP2 has a potential application in improving disease resistance in crop plants. SA and JA play important roles in the signal transduction of plant-defense systems. In general, SA is implicated in disease resistance against biotrophic pathogens, whereas JA participates in the response to necrotrophs [56]. Zhao et al. [57] and Mao et al. [58] suggested that JA- and ET-dependent pathways were induced by S. sclerotiorum infection in oilseed rape. However, a study based on the Arabidopsis mutant suggested that, in addition to the JA/ET-
mediated defense response, the SA-mediated defense response is also involved in defense against S. sclerotiorum [59]. In our current study, the expression level of PR-1 was elevated, whereas PDF1.2 transcript levels did not increase in the LJAMP2 lines when infected with S. sclerotiorum (Fig. 5), suggesting that LJAMP2 overexpression induced by S. sclerotiorum might be mediated by the SA-dependent pathway. Thus, it is an oversimplification to conclude strict separation of defenses against necrotrophic and biotrophic pathogens via SA and JA/ET signaling, respectively, based on these studies on S. sclerotiorum [60,61]. SA has a close relationship with H2O2 in plant disease resistance. Several studies have shown that SA may be related to the production of H2O2 [62e64]. The role of SA in plants may be to increase the H2O2 content through catalase inhibition [65]. The exogenous application of SA in the transgenic lines induced a significantly greater accumulation of H2O2 compared with the non-transformed plants (Fig. 6). This is consistent with our results in which the SAmediated signaling pathway was enhanced in LJAMP2 transgenic plants. Therefore, the overexpression of the LJAMP2 gene in oilseed rape in our study may provide new data on the relationship between AMPs and the disease resistance signaling pathway of the plant-defense system. Acknowledgments The authors thank Dr. Yourong Cai (Southwest University, Chongqing, China) for providing fungal stain S. sclerotiorum. This work was supported by the National Natural Science Foundation of China (31171620), the National Key Project for Research on Transgenic Plant (2011ZX08010-003), the program for New Century Excellent Talents in University (NCET-11-0700) and The Research Fund for the Doctoral Program of Higher Education (20110182110004). References
Fig. 6. H2O2 accumulation in oilseed rape plants overexpressing LJAMP2. In situ detection of H2O2 was performed by using 3, 3-diaminobenzidine staining in the untransformed wild-type (WT) control and transgenic line (#6-5).
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